Technical Reference #300

300.

Activation function-1 domain of androgen receptor contributes to the interaction between two distinct subnuclear compartments Kiminobu Goto; Yue Zhaoa; Masayuki Saito; Arihiro Tomura; Hidetaka Morinaga a; Masatoshi Nomuraa; Taijiro Okabea; Toshihiko Yanase; Ryoichi Takayanagi; Hajime Nawata, Kyushu University, Journal of Steroid Biochemistry & Molecular Biology, 85(300), (2003)

Abstract:
The nucleus contains different sets of functional compartments often called “speckles”. The splicing factor compartment (SFC) hasbeen speculated to consist of SFs and transcription factors which thus make transcription-splicing coupling possible at the

Keywords:
Activation function-1; androgen receptor; splicing factor compartments`

Materials & Methods:
pEYFP-ANT-1 and pANT-myc were constructed by inserting the ANT-1 cDNA into Kpn I and Sma I sites of pEYFP-C2 (Clontech) and Kpn I and Not I sites of pcDNA3-myc-his respectively. The reporter plasmid pMMTV-luc containing the luciferase gene driven by mouse mammary tumor virus LTR harboring hormone response element for both AR and glucocorticoid receptor (GR) have been described previously. Expression plasmids for human estrogen receptor (ER ) (pSG5-ER and a reporter plasmid for ER harboring three copies of estrogen response elements and pGEX-4T-ER(29–180 aa) pGEX-4T-ER(AF-2) were kindly provided by Dr. Shigeaki Kato (Institute of Molecular and Cellular Biosciences University of Tokyo Japan). 2.2. Isolation of ANT-1 by swapped yeast two-hybrid screening and mRNA analysis MatchMaker Plus (Clontech) was used for the yeast two-hybrid screening. Total RNA was isolated from primary-cultured human skin fibroblasts followed by a poly(A) RNA fractionation. cDNA library was constructed using TimeSaver cDNA Synthesis Kit (Amersham Pharmacia Biotech) with random primers (Amersham Pharmacia Biotech) and was inserted into pLexA-BD included in the kit. A cDNA fragment for bait encoding NTD (1–532 aa residue) of human AR was ligated in frame into pB42-AD thus creating the pB42-AD-AF-1 expressing for AR-AF-1 fused to GAL1 activation domain. Yeast EGY48 strain was transformed with the pLexA-BD carrying cDNA libraries and with the pB42-AD-AF-1 according to the manufacturer’s protocol then transformants were selected for growth on an appropriate nutrition medium. Positive candidate plasmids for AR-AF-1 binding proteins were recovered from the yeast and the nucleotide sequences were determined using the ABI PRISM 377 DNA Sequencer (Perkin-Elmer). The specificity of interaction was further confirmed by a liquid galactosidase assay. To obtain full-length ANT-1 cDNA the partial cDNA fragment encoding 78–495 aa residues of ANT-1 obtained by the two-hybrid screening was 32P-labeled as a probe for the screening of human prostate cDNA library carried by a gt10 phage vector (Clontech). The full-length ANT-1 cDNA fragment was ligated into pcDNA3 (Invitrogen) to create pcDNA3-ANT. For the Northern blot analysis MTN blots were purchased from Clontech. 2.3. Cell culture transient transfection reporter assay COS-7 and the prostatic cancer cells ALVA-41 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The cells were transiently transfected using a Superfect Transfection Kit (Life Technologies Inc.). The total amounts of transfected plasmid DNA were kept constant by adding pcDNA3 vector plasmid. At 16 h post-transfection the cells were rinsed and then were fed with medium containing 10% charcoal-stripped fetal calf serum with or without various steroid hormones. After an additional 18 h the cells were harvested and assayed for luciferase activities using the Dual-Luciferase Reporter Assay System (Promega). 2.4. Protein–protein interaction For immunocoprecipitation analysis COS-7 cells were transfected with plasmids expressing for myc-tagged ANT-1 and full-length or truncated AR and were maintained with or without 10−8M of dihydrotestosterone (DHT). Whole cell lysates were prepared by lysing cells in a buffer (1.0% Nonidet P-40 50mM Tris–Cl 150mM NaCl 1mM dithiothreitol one tablet of protease inhibitor cocktail). In one experiment nuclear lysates were prepared. The lysates were incubated at 4 ◦C for 1 h with the antibody raised against c-myc (Santa Cruz Biotechnology) in immunoprecipitation (IP) buffer (0.5% Nonidet P-40 1mM EDTA 50mM Tris–Cl 200mM NaCl 1mM dithiothreitol one tablet of protease inhibitor cocktail) and then were further incubated with protein-A Sepharose beads (Pharmacia) at 4 ◦C for 2 h. After being washed the pellets were suspended in an SDS-PAGE sample buffer. The proteins were separated on SDS-PAGE transferred to nitrocellulose filter and then were subjected to a Western blot analysis using antibody against AR (N-20) (Santa Cruz Biotechnology) for the detection of full-length or NTD fragment of AR or using antibody C-19 (Santa Cruz Biotechnology) for the detection of C-terminal fragment of AR. 2.5. Microscopy and imaging analysis The cells were divided into 35mm glass-bottom dishes (MatTek Corporation) and then were transfected with 0.5 g of pAR-CFP and pANT-1-YFP using 2.5 l per dish of Superfect reagents (QIAGEN). Six to eighteen hours post-transfection the culture medium was replaced with a fresh DMEM containing 10−8M DHT. Confocal microscopy was performed essentially as previously described. In brief 1 h after adding DHT the cells were scanned using Leica TCS-SP system (Leica Microsystems Heidelberg Germany). The cells were imaged for yellow or cyan fluorescence by excitation with the 514 and 450 nm line respectively from an argon laser. The emissions were viewed through either a 530–590 nm band pass filter for YFP or a 470–500 nm band pass filter for CFP. The nuclei were stained with Hoechst 33342 (2 g/ml) and were imaged by excitation with the 350 nm line from a UV laser and the emission was viewed through a 400–450 nm band pass filter. A series of 30–50 images were collected for each single nucleus. In each plane the cyan yellow and ultraviolet fluorescence were consecutively collected using the serial scanning methods equipped in Leica TCS-SP system. Three-dimensional image reconstruction was performed by either using the 3D analysis TRI Graphics Program software package (Ratoc System Engineering Tokyo)or using the deconvolution method (nearest neighbors).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
COS-7, ALVA-41